The Role of L-carnitine in Treatment of a Murine …The Role of L-carnitine in Treatment of a Murine Model of Asthma Nevin Uzuner욏웬,Salih Kavukçu욏,Osman Yılmaz욐,Selmin 썤Ozkal욑,

The Role of L-carnitine in Treatment of a Murine

Model of Asthma

Nevin Uzuner , Salih Kavukçu , Osman Yılmaz , Selmin Ozkal,

Huray･

f

lekel , Ozkan Karaman , Alper Soylu , and Aydanur Kargı

Departments of Pediatrics, Physiology, Pathology and Biochemistry,Dokuz Eylul University Faculty of Medicine, Izmir, Turkey

Leukotrienes, one of the mediators of inflammation in asthma, have a strong bronchoconstrictive

effect. L-carnitine has been reported to influence respiratory functions. It has also been reported

that L-carnitine inhibits leukotriene synthesis. To evaluate the effects of L-carnitine on oxygen

saturation, urine leukotriene E4 levels and lung histopathology in a murine model of asthma, high

IgE responder BALB/c mice (n＝24) were systemically sensitized to ovalbumin and chronically

challenged with low particle mass concentrations of aerosolized ovalbumin, and then they were

divided into 3 groups(study groups A, B, and C)each including eight mice. After methacholine-induced bronchoconstriction, the mice in groups A and B were given intraperitoneal L-carnitine(250 and 125 mg/kg, respectively), while the mice in group C were given placebo. Oxygen saturation

of the mice was measured by pulse oxymeter before and after methacholine and after L-carnitine/placebo application. In addition, urine leukotriene E4 levels were measured before asthma develop-ment, and 24-h after L-carnitine injection in asthmatic mice. Inflammation in the lung tissues of

the sacrificed animals was scored histopathologically to determine the effect o

g

L-carnitine on tissue

level. A control group of non-sensitized mice (n＝8) treated with placebo only was used for

comparison of urine leukotriene E4 levels and of histopathological parameters. Oxygen saturation

of the mice in the study groups tended to decrease after methacholine and to improve after

L-carnitine injection, although these changes were not significant at all time points. Urine

leukotriene E4 levels of all 3 study groups increased significantly after asthma development. The rate

of increment was smallest in the group given the highest L-carnitine dose(group A). Inflammation

at the tissue level was also mildest in group A, and severest in the group that was not given carnitine(group C). All of the study

s

roups and the control group differed significantly with respect to

inflammation scores. In conclusion, L-carnitine improved oxygen saturation, and decreased urine

leukotriene E4 levels and inflammation in lung tissues in the present murine model of asthma.

Key words:asthma, L-carnitine, leukotriene E4, oxygen saturation

A sthma is a chronic inflammatory di

l

ease of the

airways［1］. Various endogenous mediators play

a role in inflammation. Leukotrienes are among the

mediators which are synthesized in the bronchial mucosa

by eosinophi c s, basophils and mast k ells. Leu i otr s ene

s

Copyrightｃ2002 by Okayama University Medical School.

Original Article

Acta Med. Okayama, 2002

Vol. 56, No. 6, pp. 29 5-301

http://www.lib.okayama-u.ac.jp/www/acta/

Received October 26,2001;accepted July 10,2002.Corresponding author.Phone:＋90-232-2595959;Fax:＋90-232-2599723

E-mail:nuzuner＠deu.edu.tr(N.Uzuner)

play a very important role in asthma pathogenesis, and

are involved in eosinophilic inflammation, bronchocon-striction and edema formation［2］.L-carnitine has been reported to improve the obstruc-

tive findings in pulmonary function tests in children

undergoing chronic hemodialysis［3］. It has also been

reported that L-carnitine decreases leukotriene synthesis

by inactivation of lipoxygenase in hemodialysis patients［4］. We previously evaluated the bronchodilator effect of

L-carnitine in tracheal and bronchial smooth muscle of

Guinea pigs and in human bronchial smooth muscle in

vitro, but observed no significant effects. However, since

these tissues were taken from healthy subjects without

asthma, this lack of significance does not exclude a

possible relation between asthma and L-carnitine［5］.The aim of this study was to evaluate the effects of

L-carnitine on arterial oxygen saturation (SaO), urine

leukotriene E4(LTE4)levels, and lung histopathology in

a murine model of asthma.

Materials and Methods

BALB/c mice with

87 homogeneity were used for the experiment. The

mice were 8- to 10-weeks-old and weighed 28-30 g.They were kept in hygienic macrolane cages and in

air-conditioned rooms under a 12-h light/12-h dark cycle.Thirty-two mice were divided into 4 groups, the study

groups A, B, C and the control group, each including

eight mice. The study was approved by the local ethical

committee(00/09-02).

BALB/c mice are high IgE responders to ovalbumin［6］.The mice in the study groups A, B, and C were

sensitized by intraperitoneal injection of 10μg of alum

precipitated chicken egg ovalbumin (grade V, 98

pure, Sigma, St. Louis, MO, USA)21 and 7 days

before inhalational exposure. The mice in the control

group were given saline solution by the same route and

dosage.The mice in the study groups were then exposed to

aerosolized ovalbumin for 30 min per day on 3 days of the

week up to 8 weeks［6］. Exposures were carried out in

a whole body inhalation exposure system. Temperature

and relative humidity were maintained at 20-25°C and 40-60 , respectively. A solution of 2.5 ovalbumin in

normal saline was aerosolized by delivery of compressed

air to a sidestream jet nebulizer and injected into a

chamber. The aerosol generated by this nebulizer com-prised 80 particles with a diameter of 4μm.Particle concentration was maintained in the range of 10-20 mg/m. The mice in the control group were exposed

to saline inhalation by the same system.

Mice with experimentally induced chronic

asthma in the study groups A, B and C were given 3

doses of methacholine (at 6.25-12.5 and 25.0 mg/ml

concentrations)for 3 min by the same system used for

administration of aerosolized ovalbumin［7］. The time

interval between the methacholine doses was 1h. SaOwas measured by pulse oxymeter just before(0 min)and

5 min after each dose of methacholine. After that, while

intraperitoneal L-carnitine was given at 250 mg/kg and

125 mg/kg to the mice in groups A and B, respectively,the mice in group C were not given L-carnitine. Instead,isotonic saline as placebo was injected by the same route

to the animals in group C［8］. A third measurement of

SaO was also obtained 15 min after L-carnitine/placebo

administration in these 3 study groups.The control group of mice without asthma were not

treated with either methacholine or carnitine. Thus, the

responses to methacholine and carnitine, as determined

by SaO, were compared among the 3 study groups of

asthmatic mice.

Twenty four-hour urine samples of the asthmatic mice in

the study groups were collected twice, once before the

induction of asthma and once after L-carnitine administra-tion, by using metabolic cages. Similarly, 24-h urine

samples of the control mice were collected at the begin-ning of the study and just before sacrifice. The samples

were separated into 2 aliquots. The first aliquot was used

to measure urinary creatinine levels as the concentration

of picric acid complex in alkaline media using a routine

spectrophotometric method (Hitachi 911 autoanalyzer;Hitachi, Tokyo, Japan). The second aliquot was sup-plemented with 6N hydrochloric acid in order to acidify

the urine to pH 3 for measurement of LTE4, then stored

at－70°C till analysis. Urine samples were purified using

C reverse phase cartridges (500 mg/8.0 ml) (Altech

Associates, Inc.2051 Waukegan Road, Deerfield, IL,USA). During purification the samples were traced with

tritium-labeled LTE4(［ H］-LTE4)(Dupont-NE, Bos-ton, MA, USA)with the aim of calculating the recovery

factor for LTE4［9, 10］. In purified samples LTE4

levels were determined using an LTE4 EIA (Enzyme

Uzuner et al. Acta Med. Okayama Vol. 56, No. 6 29 6

Immunoassay) kit (Cayman Chemical Company, Ann

Arbor, MI, USA). The results were expressed as pg

LTE4/mg creatinine in urine.The mice in

the study groups were sacrificed 24-h after the last dose

of carnitine/placebo. Control mice were sacrificed at the

same time as the experimental mice. The trachea and

lungs of the mice were inflated with 10 buffered forma-lin, and after fixation overnight, a horizontal slice was

obtained from the left lung. The slices were embedded in

paraffin, sectioned into 5-μm thick sections, and stained

with hematoxylin-eosin. The degree of bronchial inflam-mation was evaluated semi-quantitatively using scores of

0-3 to indicate no, mild, moderate, and severe inflam-mation［6, 11］. In addition, the distribution and inten-sity of the following findings were recorded:1) bron-choconstriction (epithelial shedding or undulation of the

nuclei of bronchial epithelial cells);2) increase in the

number of goblet cells;3)infiltration of inflammatory cells

and fibrin from vessels into the mucosal and submucosal

area of the bronchus and peribronchial interstitium;and

4)hypertrophy and thickening of the smooth muscle cell

layer. A score of 0 indicated normal histology and a score

of 3 indicated the greatest degree of alteration from

normal.

Paired t-test(for n

6)and Wilcoxon’s signed rank test(for n 5)were used

for the comparison of SaO before and after various

methacholine dosages and after L-carnitine administration.Levels of significance was accepted as＜0.05. Urinary

LTE4 levels were compared within each group at the

beginning and at the end of the study by paired t-test(n

6)and Wilcoxon’s signed rank tests (n 5). Mean

increase in LTE4 level was analyzed by one way

ANOVA and post-hoc Duncan tests. Histopathological

scores were compared among the four groups by Kruskal-

Wallis test. Advanced analyses of the groups were

performed by Mann-Whitney U test after Bonferroni

correction.

Results

A total of 4 mice, 1 in group A and 3 in group B,died due to hypoxia during the study period.

SaO measured before and after methacholine administra-tion in all 3 study groups and after L-carnitine administra-tion in groups A and B or placebo in group C are shown

in Tables 1 to 3. SaO was found to decrease after all

methacholine doses in all 3 groups. However, the

decrease in SaO was not always statistically significant.On the other hand, SaO had a tendency to increase after

L-carnitine administration, and this increase was signifi-cant at the 250 mg/kg dose(in group A).SaO decreased significantly after administration of a

6.25 mg/ml dose of methacholine(P＝0.014), while it

increased significantly after administration of 250 mg/kg

L-carnitine in group A(P＝0.027)(Table 1). Similarly,administration of 25 mg/ml methacholine or 250 mg/kg

L-carnitine led to a significant decrease(P＝0.014)and a

significant increase (P＝0.012) in SaO, respectively(Table 3).In the case of group B, 6.25 mg/ml of methacholine

decreased SaO significantly(P＝0.007)and 125 mg/kg

of L-carnitine increased SaO, but not significantly(Table

1). Similar results were obtained in this group when the

mice were given 12.5 and 25 mg/ml of methacholine(Tables 2 and 3).In group C, SaO decreased at all doses of metha-

choline, but the decrease was only significant after a dose

of 6.25 mg/ml(Table 1, P＝0.034). However, SaOdid not tend to increase after placebo administration

L-carnitine in Asthma December 2002

Table 1 Oxygen saturation of the mice in groups A, B and C before and after methacholine(6.25 mg/ml)and after L-carnitine/placebo

administration

SaO0 min

(before methacholine)5 min

after methacholine

15 min after

L-carnitine/placebo

Group A(n＝7) 86.87±10.43 64.25±19.08 85.37±14.84Group B(n＝5) 89.57± 5.38 65.71±11.26 71.16± 9.06

Group C(n＝8) 86.85± 6.36 81.28± 4.57 80.71± 5.36

, Group A and B were given 250 and 125 mg/kg L-carnitine, respectively;group C was given placebo. , SaO significantly decreased after

methacholine(P＝0.014); , SaO significantly increased after L-carnitine(P＝0.027); , SaO significantly decreased after methacholine(P＝0.007); , SaO significantly decreased after methacholine(P＝0.034).

29 7

(Tables 1-3).Intragroup

evaluation revealed significantly higher levels of urinary

LTE4 after asthma development in all study groups(Table 4). After that, the difference between the post-and

pre-asthmatic urinary LTE4 levels was calculated for each

group. The mean differences were 12.11±8.49, 17.32±11.02, 44.32±06.12 and 8.59±2.59 pg LTE4/mg

creatinine in groups A, B, C and the control group,respectively. When all 4 groups were analyzed with

respect to these differences, group C had statistically

significantly higher levels compared to the others (P＝0.000).

The mean

scores with respect to bronchial inflammation were 1.14±0.37, 1.75±0.88, 1.77±0.75 and 0.33±0.5 in groups

A, B, C and the control group, respectively. The

intergroup variation was significant(P＝0.005). In post-hoc analyses, the control group was found to be

significantly different from groups B and C, but not from

group A(P＝0.008, P＝0.008 and P＝0.035, respec-tively).Lung sections in group A mice revealed mild

inflammation in peribronchial and perivascular areas. In

addition, there was mild constriction and thickening of the

walls in a small number of bronchi (Fig. 1). Vascular

Table 2 Oxygen saturation of the mice in groups A, B and C before and after methacholine(12.5 mg/ml)and after L-carnitine/placebo

administration

SaO0 min

(before methacholine)5 min

after methacholine

15 min after

L-carnitine/placebo

Group A(n＝7) 85.57±12.36 73.57±15.67 85.10± 3.60

Group B(n＝5) 87.00± 9.29 78.33± 9.77 84.20±14.72

Group C(n＝8) 78.71±23.22 68.42±30.81 70.03±15.52

, Group A and B were given 250 and 125 mg/kg L-carnitine, respectively;group C was given placebo. , SaO significantly decreased after

methacholine(P＝0.026).

Table 3 Oxygen saturation of the mice in groups A, B and C before and after methacholine (25 mg/ml)and after L-carnitine/placebo

administration

SaO0 min

(before methacholine)5 min

after methacholine

15 min after

L-carnitine/placebo

Group A(n＝7) 85.01± 6.09 65.14±19.78 84.57±11.70Group B(n＝5) 72.50± 9.29 75.75±10.78 79.25± 4.11

Group C(n＝8) 82.00±12.02 74.47±10.65 72.33± 9.48

, Group A and B were given 250 and 125 mg/kg L-carnitine, respectively;group C was given placebo. , SaO significantly decreased after

methacholine(P＝0.014); , SaO significantly increased after L-carnitine(P＝0.012).

Table 4 Pre-and post-asthmatic(after a 25 mg/ml dose of methacholine)urinary leukotriene E4 levels in mice

Urine Leukotriene E4 Levels (Mean±SD)(pg LTE4/mg creatinine)

Groups Pre asthmatic Post asthmatic(after L-carnitine)

P

A(n＝7) 0.64±0.52 12.75± 8.80 0.018

B(n＝5) 3.53±2.86 20.85±11.89 0.018

C(n＝8) 10.71±5.03 55.03±11.15 0.028

Control Group (n＝8) 2.76±2.50 11.36± 3.73 0.042

, The mice in group A were given 250 mg/kg L-carnitine, and those in group B were given 125 mg/kg of L-carnitine. The mice in group C

and the control group were not given L-carnitine. , Urine LTE4 levels were studied at the beginning and end of the study in control group.

Uzuner et al. Acta Med. Okayama Vol. 56, No. 6 29 8

Fig.1 Peribronchial mild inflammation(H&E×100, group A).

Fig.3 Peribronchial intense inflammation and thickening of the

bronchial walls (H&E×40, group C).

Fig.5 Thickening of and fibrin deposition in the vessel walls, and

perivascular mild inflammation(H&E×400, group C).

Fig.2 Peribronchial moderate inflammation(H&E×100, group B).

Fig.6 Mild focal peribronchial and perivascular inflammation(H&E×40, control group).

Fig. 4 Bronchoconstriction and mild peribronchial inflammation(H&E×40, group C).

29 9 L-carnitine in Asthma December 2002

changes like thickening of vessel walls and fibrin deposi-tion were mild and present in a small number of vessels.Bronchial inflammation was moderate in group B mice(Fig. 2). Bronchoconstriction and perivascular inflam-mation were also more pronounced in group B than in

group A mice. Thickening of bronchial and vascular walls

were not different between these 2 groups.Group C mice showed severe peribronchial

inflammation, and the other histopathological changes,including bronchoconstriction (Fig.3)and perivascular

inflammation(Fig.4), were also more pronounced in this

group. In addition, the vessel walls of group C mice

showed thickening and fibrin deposition(Fig.5). There

were no histopathologic changes other than mild focal

inflammation in the lung tissues of the control mice(Fig.6). None of the groups showed any increase in the

number of goblet cells.

Discussion

Leukotrienes, a product of the lipooxygenase path-way, bind to the leukotrien receptors present in bronchial

smooth muscle and are found in increased concentrations

in the bronchoalveolar fluid of asthmatic patients［12, 13,14］.In hemodialysis patients, the metabolism of ara-

chidonic acid has been shown to shift from the cycloox-ygenase to the lipoxygenase pathway［4］. When L-carnitine was administered to children undergoing

hemodialysis, the obstructive pattern in respiratory func-tions was shown to improve［3］. Carnitine was demon-strated to prevent bronchospasm due to Cys LT by

blocking the lipoxygenase pathway in these patients.L-carnitine causes partial restoration of the depleted

essential fatty acids(linoleic and linolenic acids)observed

in untreated dialysis patients［4］. Although the mecha-nism by which carnitine corrects these abnormalities is

unclear, it has been shown that dietary sources of

alpha-linolenic acid may have the capacity to inhibit the

generation of leukotrienes by leucocytes in patients with

asthma［15］. Thus, carnitine might act on leukotriene

metabolism by altering the ratio of essential fatty acids.In a previous in vitro study, we showed that L-

carnitine did not affect bronchoconstriction caused by

methacholine in guinea pig tracheal and bronchial smooth

muscle, or in human bronchial smooth muscle. However,since there was no asthma pathology in the smooth

muscles in that study, it was speculated that L-carnitine

did not affect the bronchial smooth muscles under

physiologic conditions［5］.We performed this study to investigate the effects of

L-carnitine on acute attacks in a chronic asthma model in

mice, and whether leukotriene synthesis inhibition was

involved in these effects, if any. In this study, acute

attacks were induced by methacholine after the chronic

asthma model was developed, and the effect of L-carnitine

on SaO, as an indirect indicator of bronchoconstriction,during acute attacks was observed. In addition, the role

of L-carnitine on leukotriene synthesis was evaluated by

measuring urinary LTE4 levels. More over, the effect of

L-carnitine on acute attacks in chronic asthma was inves-tigated at the tissue level.Post-methacholine SaO decreased, although not

significantly at all times, in the study groups(Tables 1-3). This finding indicate indirectly that there is bronchial

hyperreactivity and inflammation in these animals with

acute bronchospasm. After L-carnitine administration,SaO increased significantly in group A(given 250 mg/kg

L-carnitine), but increased insignificantly in group B(given 125 mg/kg L-carnitine). These findings show that

higher doses of L-carnitine had a more pronounced

bronchodilator effect in this chronic murine asthma model.LTE4 is the major metabolite of leukotriene metabo-

lism and is excreted in urine. While other leukotriene

metabolites are rapidly metabolized in vivo, LTE4 is

more stable. Thus, LTE4 levels are often used as a

marker of in vivo leukotriene production［16］, as they

were here. We measured urine LTE4 levels before the

development of asthma and after L-carnitine administra-tion in asthmatic mice. Ideally, we would also have

measured LTE4 levels in asthmatic mice before carnitine

treatment. Unfortunately, we were unable able to do this

due to the limited materials for LTE4 measurement.However, we found significantly increased levels of urine

LTE4 even after L-carnitine administration in asthmatic

mice in all study groups. We then calculated the mean

differences between the post-and pre-asthmatic urine

LTE4 levels for each group. The control group showed

the lowest difference (8.59±2.59 pg LTE4/mg creat-inine), followed in increasing order by group A(12.11±8.49), group B (17.32±11.02)and group C (44.32±06.12). These findings indicated that L-carnitine treat-ment decreased urinary LTE4 excretion, and suggested

that L-carnitine might be effective in reducing the

inflammatory process in asthma.There were statistically significant differences among

Uzuner et al. Acta Med. Okayama Vol. 56, No. 6 300

the 4 groups with respect to the intensity of bronchial

inflammation. The lowest inflammatory scores were in the

control group (0.33±0.5) and group A (1.14±0.37).These 2 groups did not differ significantly. On the other

hand, the scores in group B (1.75±0.88)and group C(1.77±0.75)were significantly different from those in the

control group. Peribronchial and perivascular inflam-mation was significantly lower in group A than in group

B or group C, which had the highest scores. As such,bronchoconstriction and thickening of bronchial walls

were prominent in group C, but were present in only a

small number of bronchi in group A. Likewise, thicken-ing of vessel walls and fibrin deposition were mild in

group A and extensive in group C. These findings show

that the histopathological changes of asthma were

attenuated in the mice given L-carnitine(groups A and B),and were particularly small in group A, which was given

the higher dose of L-carnitine. These findings, in turn,support our hypothesis that L-carnitine has bronchodilator

and anti-inflammatory properties.In conclusion, intraperitoneal administration followed

by aerosolized ovalbumin resulted in asthma development

in BALB/c mice, and while methacholine administration

decreased SaO, L-carnitine administration increased

SaO in these asthmatic mice. In addition, urinary LTE4

levels and histopathological injury scores were lower in

the asthmatic mice given L-carnitine. These results

suggest that L-carnitine might have a role in the treatment

of experimentally induced asthma in mice. Additional,larger-scale studies will be needed to confirm the

effectiveness of L-carnitine on inhibition of leukotrienes.

Acknowledgements. We thank very much to Çarmosan-Milupa Company for

their financial support in this syudy.

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301 L-carnitine in Asthma December 2002